Integration of network pharmacology, transcriptomics and single-cell sequencing to explore the effect of Rougan Keli in alleviating liver cirrhosis.

Abstract

Background: The progression of liver cirrhosis leads to severe complications, significantly threatening the survival and prognosis of patients. Rou gan keli (Rgkl), a herbal formula derived from classical prescriptions, has used clinically over two decades and has good efficacy. However, its molecular mechanisms and active components remain undefined.

Purpose: Exploring the molecular mechanisms of Rgkl in alleviating liver cirrhosis.

Methods: CCl₄-induced liver cirrhosis mice models were established. Liver stiffness and intrahepatic blood flow velocity were assessed using imaging. Serum ALT, AST, HA, and histopathology were analyzed. Hepatic stellate cells (HSCs) activation, liver sinusoidal endothelial cells (LSECs) fenestration, and angiogenesis were evaluated using immunohistochemistry and scanning electron microscopy. UPLC-Q-TOF-MS/MS and network pharmacology identified active components. Transcriptomics and single-cell sequencing identified key targets and pathways, validated via WB, immunofluorescence, and molecular docking.

Results: Rgkl significantly reduced Liver stiffness and collagen deposition while increasing intrahepatic blood flow velocity in cirrhotic mice. Serum ALT, AST, and HA were markedly decreased. Rgkl inhibited α-SMA expression in HSC and downregulated pathological angiogenesis by reducing VEGF and CD34 expression. Additionally, Rgkl enhanced eNOS expression and preserved sinusoidal fenestration in LSEC. Furthermore, Rgkl ameliorated liver cirrhosis by modulating LSEC metabolic functions via the CD36/PPAR/CPT-1 pathway and suppressing HSC activation through the RhoA/ROCK/YAP and PI3K/AKT/NF-κB pathways. Eighteen active components, such as Levistilide A and Quercetin, were strongly correlated with the amelioration of liver cirrhosis.

Conclusions: Rgkl significantly attenuated hepatic injury and fibrosis. Mechanistically, Rgkl modulated LSEC lipid metabolism and phenotypic regulation, and suppressed HSC contraction and activation. Key active components contributing to these effects included Paeonilactone C, Levistilide A, and Quercetin.